Skip to main content

Advertisement

SpringerLink
Log in

Expression and distribution of transcription factor CCAAT/enhancer-binding protein in the central nervous system of Lymnaea stagnalis

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

The transcription factor, CCAAT/enhancer-binding protein (C/EBP), is involved in important physiological processes, such as cellular proliferation and differentiation, homeostasis, and higher-order functions of the brain. In the present study, we investigated the distribution of mRNA and protein of C/EBP in the central nervous system of the pond snail, Lymnaea stagnalis, by in situ hybridization and immunohistochemistry. Specificity of the anti-mammalian C/EBP antibody against Lymnaea C/EBP (LymC/EBP) was confirmed by combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis or isoelectric focusing and immunoblotting. Cells positive for in situ hybridization were immunoreactive for LymC/EBP in all 11 ganglia. The motoneurons (B1, B2, B4, and B4 clusters) in the buccal ganglia and interneurons (cerebral giant cell, CGC) in the cerebral ganglia were positive for in situ hybridization and were immunopositive. In the pedal ganglion, the right pedal dorsal 1 (RPeD1), pedal A, and pedal C clusters exhibited positive signals of in situ hybridization and immunohistochemistry for LymC/EBP. CGC and RPeD1 are key neurons for associative learning. In addition, the neuropeptidergic cells in the cerebral, pleural, parietal, and visceral ganglia were positive for in situ hybridization and immunoreactive. Interestingly, although the cytoplasm of almost all immunopositive cells was stained, some neuropeptidergic cells located in the light parietal and visceral ganglia exhibited immunoreactivity in nuclei. Our results suggest that LymC/EBP is involved in learning and memory and in the expression and/or secretion of neuropeptides in Lymnaea.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Alberini CM, Ghirardi M, Metz R, Kandel ER (1994) C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia. Cell 76:1099–1114

    Article  CAS  PubMed  Google Scholar 

  • Bartsch D, Ghirardi M, Skehel PA, Karl KA, Herder SP, Chen M, Bailey CH, Kandel ER (1995) Aplysia CREB2 represses long-term facilitation: relief of repression converts transient facilitation into long-term functional and structural change. Cell 83:979–992

    Article  CAS  PubMed  Google Scholar 

  • Bartsch D, Casadio A, Karl KA, Serodio P, Kandel ER (1998) CREB1 encodes a nuclear activator, a repressor, and a cytoplasmic modulator that form a regulatory unit critical for long-term facilitation. Cell 95:211–223

    Article  CAS  PubMed  Google Scholar 

  • Bartsch D, Ghirardi M, Casadio A, Giustetto M, Karl KA, Zhu H, Kandel ER (2000) Enhancement of memory-related long-term facilitation by ApAF, a novel transcription factor that acts downstream from both CREB1 and CREB2. Cell 103:595–608

    Article  CAS  PubMed  Google Scholar 

  • Billiard J, Umayahara Y, Wiren K, Centrella M, McCarthy TL, Rotwein P (2001) Regulated nuclear-cytoplasmic localization of CCAAT/enhancer-binding protein delta in osteoblasts. J Biol Chem 276:15354–15361

    Article  CAS  PubMed  Google Scholar 

  • Boer HH, Montagne-Wajer C, Minnen J van, Ramkema M, Boer P de (1992) Functional morphology of the neuroendocrine sodium influx-stimulating peptide system of the pond snail, Lymnaea stagnalis, studied by in situ hybridization and immunocytochemistry. Cell Tissue Res 268:559–566

    CAS  PubMed  Google Scholar 

  • Boer HH, Montagne-Wajer C, Smith FG, Parish DC, Ramkema MD, Hoek RM, Minnen J van, Benjamin PR (1994) Functional morphology of the light yellow cell and yellow cell (sodium influx-stimulating peptide) neuroendocrine systems of the pond snail Lymnaea stagnalis. Cell Tissue Res 275:361–368

    CAS  PubMed  Google Scholar 

  • Cortés-Canteli M, Wagner M, Ansorge W, Pérez-Castillo A (2004) Microarray analysis supports a role for CCAAT/enhancer-binding protein-β in brain injury. J Biol Chem 279:14409–14417

    Article  PubMed  Google Scholar 

  • Croll RP, Chiasson BJ (1989) Postembryonic development of serotonin-like immunoreactivity in the central nervous system of the snail, Lymnaea stagnalis. J Comp Neurol 280:122–142

    CAS  PubMed  Google Scholar 

  • Hatakeyama D, Ito E (1999) Three-dimensional reconstruction and the mapping of serotonin-like immunoreactive neurons in the central nervous system of the pond snail, Lymnaea stagnalis, with the confocal laser scanning microscope. Bioimages 7:1–12

    CAS  Google Scholar 

  • Hatakeyama D, Ito E (2000) Distribution and developmental changes in GABA-like immunoreactive neurons in the central nervous system of pond snail, Lymnaea stagnalis. J Comp Neurol 418:310–322

    Article  CAS  PubMed  Google Scholar 

  • Hatakeyama D, Ito I, Kojima S, Fujito Y, Ito E (2000) Complement receptor 3-like immunoreactivity in the light green cells and the canopy cells of the pond snail, Lymnaea stagnalis. Brain Res 865:102–106

    Article  CAS  PubMed  Google Scholar 

  • Hatakeyama D, Ito I, Ito E (2001) Complement receptor 3-like immunoreactivity in the superior and inferior tentacles of terrestrial slug, Limax marginatus. Zool Sci 18:5–10

    CAS  Google Scholar 

  • Hatakeyama D, Sadamoto H, Ito E (2004a) Real-time quantitative RT-PCR method for estimation of mRNA level of CCAAT/enhancer binding protein in the central nervous system of Lymnaea stagnalis. Acta Biol Hung 55:157–161

    Article  CAS  PubMed  Google Scholar 

  • Hatakeyama D, Inamura S, Ito E, Sakakibara M, Nelson TJ, Alkon DL (2004b) Calexcitin-like immunoreactivity in the pond snail Lymnaea stagnalis. Neurosci Res Commun 35:32–40

    Google Scholar 

  • Hattar S, Lyons LC, Eskin A (2002) Circadian regulation of a transcription factor, ApC/EBP, in the eye of Aplysia californica. J Neurochem 83:1401–1411

    Article  CAS  PubMed  Google Scholar 

  • Hermann PM, Lange RP de, Pieneman AW, Maat A ter, Jansen RF (1997) Role of neuropeptides encoded on CDCH-1 gene in the organization of egg-laying behavior in the pond snail, Lymnaea stagnalis. J Neurophysiol 78:2859–2869

    CAS  PubMed  Google Scholar 

  • Huang Z, You W, Haugland RP, Paragas VB, Olson NA, Haugland RP (1993) A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ. J Histochem Cytochem 41:313–317

    CAS  PubMed  Google Scholar 

  • Ito E, Kobayashi S, Kojima S, Sadamoto H, Hatakeyama D (1999) Associative learning in the pond snail, Lymnaea stagnalis. Zool Sci 16:711–723

    Google Scholar 

  • Jimenez CR, Maat A ter, Pieneman A, Burlingame AL, Smit AB, Li KW (2004) Spatio-temporal dynamics of the egg-laying-inducing peptides during an egg-laying cycle: a semiquantitative matrix-assisted laser desorption/ionization mass spectrometry approach. J Neurochem 89:865–875

    CAS  PubMed  Google Scholar 

  • Jones NG, Kemenes I, Kemenes G, Benjamin PR (2003) A persistent cellular change in a single modulatory neuron contributes to associative long-term memory. Curr Biol 13:1064–1069

    Article  CAS  PubMed  Google Scholar 

  • Kesteren RE van, Syed NI, Munno DW, Bouwman J, Feng ZP, Geraerts WPM, Smit AB (2001) Synapse formation between central neurons requires postsynaptic expression of the MEN1 tumor suppressor gene. J Neurosci 21:RC161

    PubMed  Google Scholar 

  • Kojima S, Yamanaka M, Fujito Y, Ito E (1996) Differential neuroethological effects of aversive and appetitive reinforcing stimuli on associative learning in Lymnaea stagnalis. Zool Sci 13:803–812

    Google Scholar 

  • Kojima S, Nakamura H, Nagayama S, Fujito Y, Ito E (1997) Enhancement of an inhibitory input to the feeding central pattern generator in Lymnaea stagnalis during conditioned taste-aversion learning. Neurosci Lett 230:179–182

    Article  CAS  PubMed  Google Scholar 

  • Kojima S, Ogawa H, Kouuchi T, Nidaira T, Hosono T, Ito E (2000) Neuron-independent Ca2+ signaling in glial cells of snail’s brain. Neuroscience 100:893–900

    Article  CAS  PubMed  Google Scholar 

  • Lee JA, Kim HK, Kim KH, Han JH, Lee YS, Lim CS, Chang DJ, Kubo T, Kaang BK (2001) Overexpression of and RNA interference with the CCAAT enhancer-binding protein on long-term facilitation of Aplysia sensory to motor synapses. Learn Mem 8:220–226

    Article  PubMed  Google Scholar 

  • Lee JA, Kim H, Lee YS, Kaang BK (2003) Overexpression and RNA interference of Ap-cyclic AMP-response element binding protein-2, a repressor of long-term facilitation, in Aplysia kurodai sensory-to-motor synapses. Neurosci Lett 30:9–12

    Article  Google Scholar 

  • Lekstrom-Himes J, Xanthopoulos KG (1998) Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem 273:28545–28548

    Article  CAS  PubMed  Google Scholar 

  • Lukowiak K, Sangha S, McComb C, Varshney N, Rosenegger D, Sadamoto H, Scheibenstock A (2003) Associative learning and memory in Lymnaea stagnalis: how well do they remember? J Exp Biol 206:2097–2103

    Article  PubMed  Google Scholar 

  • Lyons SE, Shue BC, Lei L, Oates AC, Zon LI, Liu PP (2001) Molecular cloning, genetic mapping, and expression analysis of four zebrafish c/ebp genes. Gene 281:43–51

    Article  CAS  PubMed  Google Scholar 

  • Marshall J, Dolan BM, Garcia EP, Sathe S, Tang X, Mao Z, Blair LA (2003) Calcium channel and NMDA receptor activities differentially regulate nuclear C/EBPβ levels to control neuronal survival. Neuron 39:625–639

    Article  CAS  PubMed  Google Scholar 

  • Meester I, Ramkema MD, Minnen J van, Boer HH (1992) Differential expression of four genes encoding molluscan insulin-related peptides in the central nervous system of the pond snail Lymnaea stagnalis. Cell Tissue Res 269:183–188

    CAS  PubMed  Google Scholar 

  • Menard C, Hein P, Paquin A, Savelson A, Yang XM, Lederfein D, Barnabe-Heider F, Mir AA, Sterneck E, Peterson AC, Johnson PF, Vinson C, Miller FD (2002) An essential role for a MEK-C/EBP pathway during growth factor-regulated cortical neurogenesis. Neuron 36:597–610

    Article  CAS  PubMed  Google Scholar 

  • Minnen J van, Haar C van der, Raap AK, Vreugdebhil E (1988) Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization. Cell Tissue Res 251:477–484

    PubMed  Google Scholar 

  • Nakamura H, Kobayashi S, Kojima S, Urano A, Ito E (1999) PKA-dependent regulation of synaptic enhancement between a buccal motor neuron and its regulatory interneuron in Lymnaea stagnalis. Zool Sci 16:387–394

    CAS  Google Scholar 

  • Ribeiro MJ, Serfözö Z, Papp A, Kemenes I, O’Shea M, Yin JC, Benjamin PR, Kemenes G (2003) Cyclic AMP response element-binding (CREB)-like proteins in a molluscan brain: cellular localization and learning-induced phosphorylation. Eur J Neurosci 18:1223–1234

    Article  PubMed  Google Scholar 

  • Roubos, Moorer-Van Delft (1976) Morphometric in vitro analysis of the control of the activity of the neurosecretory dark green cells in the freshwater snail Lymnaea stagnalis (L.). Cell Tissue Res 174:221–231

    CAS  PubMed  Google Scholar 

  • Sadamoto H, Azami A, Ito E (2004a) The expression pattern of CREB genes in the central nervous system of the pond snail Lymnaea stagnalis. Acta Biol Hung 55:163–166

    Article  PubMed  Google Scholar 

  • Sadamoto H, Sato H, Kobayashi S, Murakami J, Aonuma H, Ando H, Fujito Y, Hamano K, Awaji M, Lukowiak K, Urano A, Ito E (2004b) CREB in the pond snail Lymnaea stagnalis cloning, gene expression, and function in identifiable neurons of the central nervous system. J Neurobiol 58:455–466

    Article  CAS  PubMed  Google Scholar 

  • Sakakibara M, Kawai R, Kobayashi S, Horikoshi T (1998) Associative learning of visual and vestibular stimuli in Lymnaea. Neurobiol Learn Mem 69:1–12

    Article  CAS  PubMed  Google Scholar 

  • Sangha S, Scheibenstock A, Lukowiak K (2003) Reconsolidation of a long-term memory in Lymnaea requires new protein and RNA synthesis and the soma of right pedal dorsal 1. J Neurosci 23:8034–8040

    CAS  PubMed  Google Scholar 

  • Schaufele F (1996) CCAAT/enhancer-binding protein a activation of the rat growth hormone promoter in pituitary progenitor GHFT1-5 cells. J Biol Chem 271:21484–21489

    CAS  PubMed  Google Scholar 

  • Smit AB, Hoek RM, Geraerts WP (1993) The isolation of a cDNA encoding a neuropeptide prohormone from the light yellow cells of Lymnaea stagnalis. Cell Mol Neurobiol 13:263–270

    CAS  PubMed  Google Scholar 

  • Smit AB, Syed NI, Schaap D, Minnen J van, Klumperman J, Kits KS, Lodder H, Schors RC van der, Elk R van, Sorgedrager B, Brejc K, Sixma TK, Geraerts WP (2001) A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature 411:261–268

    Article  CAS  PubMed  Google Scholar 

  • Soory M, Suchak A (2003) Effects of alkaline phosphatase and its inhibitor levamisole on the modulation of androgen metabolism by nicotine and minocycline in human gingival and oral periosteal fibroblasts. Arch Oral Biol 48:69–76

    Article  CAS  PubMed  Google Scholar 

  • Stephanou A, Okosi A, Knight RA, Chowdrey HS, Latchman DS (1997) C/EBP activates the human corticotropin-releasing hormone gene promoter. Mol Cell Endocrinol 134:41–50

    Article  CAS  PubMed  Google Scholar 

  • Syed NI, Winlow W (1989) Morphology and electrophysiology of neurons innervating the ciliated locomotor epithelium in Lymnaea stagnalis (L.). Comp Biochem Physiol 93A:633–644

    Article  Google Scholar 

  • Taubenfeld SM, Milekic MH, Monti B, Alberini CM (2001) The consolidation of new but not reactivated memory requires hippocampal C/EBPβ. Nat Neurosci 4:813–818

    Article  CAS  PubMed  Google Scholar 

  • Wagatsuma A, Sugai R, Chono K, Azami A, Hatakeyama D, Sadamoto H, Ito E (2004) The early snail acquires the learning. Comparison of scores for conditioned taste aversion between morning and afternoon. Acta Biol Hung 55:149–155

    Article  PubMed  Google Scholar 

  • Wendelaar Bonga SE (1970) Ultrastructure and histochemistry of neurosecretory cells and neurohaemal areas in the pond snail Lymnaea stagnalis (L.). Z Zellforsch 108:190–224

    CAS  Google Scholar 

  • Williams SC, Angerer ND, Johnson PF (1997) C/EBP proteins contain nuclear localization signals imbedded in their basic regions. Gene Expr 6:371–385

    CAS  PubMed  Google Scholar 

  • With ND de, Schors RC van der, Boer HH, Ebberink RH (1993) The sodium influx stimulating peptide of the pulmonate freshwater snail Lymnaea stagnalis. Peptides 14:783–789

    Article  PubMed  Google Scholar 

  • With ND de, Li KW, Jimenez CR, Vonk N, Dreisewerd K, Hillenkamp F, Karas M, Geraerts WP (1997) Intracellular degradation of C-peptides in molluscan neurons producing insulin-related hormones. Peptides 18:765–770

    Article  PubMed  Google Scholar 

  • Yamanaka M, Hatakeyama D, Sadamoto H, Kimura T, Ito E (2000) Development of key neurons for learning stimulates learning ability in Lymnaea stagnalis. Neurosci Lett 278:113–116

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Biological Sciences, Graduate School of Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo, 060-0810, Hokkaido, Japan

    Dai Hatakeyama & Etsuro Ito

  2. Department of Biological Science and Technology, School of High-Technology for Human Welfare, Tokai University, Nishino 317, Numazu, 410-0321, Japan

    Dai Hatakeyama & Manabu Sakakibara

  3. Department of Physiology, School of Medicine, Sapporo Medical University, Sapporo, 060-8556, Hokkaido, Japan

    Yutaka Fujito

  4. Division of Innovative Research, Creative Research Initiative “Sousei” (CRIS), North 21, West 10, Kita-ku, Sapporo, 001-0021, Hokkaido, Japan

    Etsuro Ito

Authors
  1. Dai Hatakeyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yutaka Fujito
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manabu Sakakibara
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Etsuro Ito
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dai Hatakeyama.

Additional information

This work was supported in part by a Research Fellowship for Young Scientists to D.H. (no. 5820), and a Grant-in-Aid to E.I. (nos. 14540622 and 16370033) from the Japan Society for the Promotion of Science

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hatakeyama, D., Fujito, Y., Sakakibara, M. et al. Expression and distribution of transcription factor CCAAT/enhancer-binding protein in the central nervous system of Lymnaea stagnalis. Cell Tissue Res 318, 631–641 (2004). https://doi.org/10.1007/s00441-004-0965-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-004-0965-8

Keywords

Search

Navigation